Isolation and characterization of Fusarium spp. From unhatched eggs of Caretta caretta in Tuscany (Italy).

Sea Turtle Egg Fusariosis (STEF) is a worldwide emergent fungal disease affecting eggs and causing embryos mortality in turtle's nests such as those of Caretta caretta. It is caused by a complex of species belonging to Fusarium genus, particularly those included in the Fusarium Solani Species Complex (FSSC). During the samplings carried out in summer 2020 along the Tuscany coastlines (Italy), C. caretta eggs showed clinical signs resembling those caused by STEF. A total of 32 fungal isolates were obtained from lesioned eggs whose molecular characterization allowing identifying as belonging to FSSC / Neocosmospora spp., Fusarium oxysporum Species Complex (FOSC) / F. oxysporum and Fusarium nodosum, i.e., fungal genera and speciesincluding also well-known plant pathogens. Isolates inoculated on several plant hosts did not result in any pathogenic activity but F. nodosum causing, on wheat spikes, disease symptoms.This is the first time F. nodosum has been isolated from portions of eggs showing evident signs of fungal infection. This work represents the first report of Fusarium spp. isolated from C. caretta eggs showing lesions resembling those caused by STEF on Tuscan coast thus posing a significant concern to loggerhead sea turtle conservation also in this region.

Characterization of Trichoderma species from forest ecosystems by high-throughput phenotypic microarray.

The use of beneficial organisms for the biocontrol of soil-borne pathogens in forestry is still poor explored. In this work, the nutritional demands of 10 previously selected isolates of Trichoderma for the biocontrol of forest soil-borne pathogens have been tested by Phenotype Microarray technology, to investigate about their C-source utilization and exploring the possibility to obtain a microbial consortia (SynCom), an innovative strategy for the biocontrol of plant disease. All Trichoderma isolates tested in this study showed a high spore germination percentage within 3 d and evidenced nutritional preference regardless of the species they belong to, and unrelated to their soil of origin. Results of growth curve analysis and MANOVA test revealed that all isolates assimilate a broad range of substrates, generally preferring complex compounds such as monosaccharides related compounds, nitrogen compounds, carboxylic acids and esters. No evidence of competition for nutritional resources have been observed among isolates of this study. As a result, a combination of different isolates could be proposed to obtain a SynCom useful for the practice of phytopathogen biocontrol in forestry. The addition of i-erythritol, adenosine and turanose to a growth substrate could be suggested as stimulating compounds for the growth of the selected Trichoderma isolates.

Mushroom-Based Supplements in Italy: Let's Open Pandora's Box.

Mushrooms and derivates are well known to the scientific community for having different health benefits and exhibit a wide range of pharmacological activities, including lipid-lowering, antihypertensive, antidiabetic, antimicrobic, antiallergic, anti-inflammatory, anticancer, immunomodulating, neuroprotective and osteoprotective actions. In Europe, medical mushrooms are mainly marketed in the form of food supplements as single components or combined with other nutraceuticals. In this context, the first peculiarity that distinguishes it is the safety established through the "history of consumption" that characterizes that mushroom. However, the cultivation of medicinal mushrooms on a large scale is performed mainly in China, where most of the production facilities do not have internationally recognized good manufacturing practices, despite that many European companies that sell myotherapies are supplied by Chinese manufacturers. This is particularly evident in Italy, where an arsenal of mushroom products is marketed in the form of powders and extracts not always of ascertained origin and sometimes of doubtful taxonomic identification, and thus not meeting the quality criteria required. The growing interest in mycotherapy involves a strong commitment from the scientific community to propose supplements of safe origin and genetic purity as well as to promote clinical trials to evaluate its real effects on humans. The purpose of this research is to analyze different mushroom-based dietary supplements used in medicine as monotherapy on the Italian market and to evaluate their composition and quality. The molecular identification of the sequences with those deposited in GenBank allowed for identifying 6 out of 19 samples, matching with those deposited belonging to the species indicated in the label, i.e., Lentinula edodes (samples 1, 4, 12 and 18) and Ganoderma lucidum (samples 5 and 10). Samples containing Ganoderma, labeled in the commercial product as G. lucidum, showed sequences that showed homology of 100% and 99% with G. resinaceum and G. sichuanense. An additional investigation was carried out in order to determine the active fungal ingredients, such as ergosterol, aflatoxins, heavy metals, nicotine and total glucan. The results obtained and shown in the manuscript highlight how the data were not only in line with what is expected with respect to what is indicated in the labels.

Sustainable Strategies to Counteract Mycotoxins Contamination and Cowpea Weevil in Chickpea Seeds during Post-Harvest.

Mycotoxins contamination and pest infestation of foods and feeds represent a pivotal threat for food safety and security worldwide, with crucial implications for human and animal health. Controlled atmosphere could be a sustainable strategy to reduce mycotoxins content and counteract the vitality of deleterious organisms in foodstuff. Ozone treatment (O3, 500 ppb for 30, 60 or 90 min) and high nitrogen concentration (N2, 99% for 21 consecutive days) were tested in the post-harvest management of four batches of Cicer arietinum grains to control the presence of mycotoxigenic fungi and their secondary metabolites, as well as pest (i.e., Callosobruchus maculatus) infestation. At the end of the treatment, O3 significantly decreased the incidence of Penicillium spp. (by an average of -50%, independently to the time of exposure) and reduced the patulin and aflatoxins content after 30 min (-85 and -100%, respectively). High N2 concentrations remarkably reduced mycotoxins contamination (by an average of -94%) and induced pest mortality (at 100% after 5 days of exposure). These results confirm the promising potential of O3 and N2 in post-harvest conservation strategies, leading to further investigations to evaluate the effects on the qualitative characteristics of grains.

Biological Disease Control by Beneficial (Micro)Organisms: Selected Breakthroughs in the Past 50 Years.

Biological control of plant disease by beneficial (micro)organisms is one of the main tools available to preserve plant health within the wider context of One Health and in line with the goals of the Agenda 2030 for Sustainable Development. The commercial development of biocontrol agents, together with a new perspective on the resident microbial community, all supported by innovative "omics" technologies, continues to gain in prominence in plant pathology, addressing the need to feed the increasing world population and to assure safe and secure access to food. The present review considers selected advances within the last 50 years, highlighting those that can be considered as breakthroughs for the biological control research field. Selected examples of successful biocontrol agents and strategies are reported, including the history of the progress in researching Trichoderma isolates as commercial biocontrol agents, the exploitation of mycoviruses to confer hypovirulence to plant pathogenic fungi, the role of microbial communities in the suppressiveness of soils, and evolving approaches including the establishment of synthetic microbial communities.

Genome Resources of Verticillium dahliae VdGL16: The Causal Agent of Vascular Wilt on the Invasive Species Ailanthus altissima.

Verticillium species are known as plant pathogens responsible for wilt diseases in a large variety of dicotyledon plants and crops in many parts of the world. Here we present the draft genome sequence of Verticillium dahliae Kleb. (strain VdGL16) isolated in Italy from the invasive alien species Ailanthus altissima (Mill.; commonly known as tree-of-heaven) showing Verticillium wilt symptoms. The comparison between the newly sequenced genome with those publicly available revealed candidate genes putatively involved in pathogenicity. The genome represents a new useful source for future research on Verticillium genetics and biology as well as research on novel approaches in the control of A. altissima.

Minimal impacts on the wheat microbiome when Trichoderma gamsii T6085 is applied as a biocontrol agent to manage fusarium head blight disease.

Fusarium head blight (FHB) is a major fungal disease that causes severe yield and quality loss in wheat. Biological control can be integrated with other management strategies to control FHB. For this purpose, Trichoderma gamsii strain T6085 is a potential biocontrol agent to limit the infection of F. graminearum and F. culmorum in wheat. However, the possible impacts of T. gamsii T6085 on the broader microbiome associated with the wheat plant are not currently understood. Therefore, we identified bacteria and fungi associated with different wheat tissues, including assessment of their relative abundances and dynamics in response to the application of T6085 and over time, using amplicon sequencing. Residues of the prior year's wheat crop and the current year's wheat spikes were collected at multiple time points, and kernel samples were collected at harvest. DNA was extracted from the collected wheat tissues, and amplicon sequencing was performed to profile microbiomes using 16S v4 rRNA amplicons for bacteria and ITS2 amplicons for fungi. Quantitative PCR was performed to evaluate the absolute abundances of F. graminearum and T. gamsii in different wheat tissues. Disease progression was tracked visually during the growing season, revealing that FHB severity and incidence were significantly reduced when T6085 was applied to wheat spikes at anthesis. However, treatment with T6085 did not lessen the F. graminearum abundance in wheat spikes or kernels. There were substantial changes in F. graminearum abundance over time; in crop residue, pathogen abundance was highest at the initial time point and declined over time, while in wheat spikes, pathogen abundance increased significantly over time. The predominant bacterial taxa in wheat spikes and kernels were Pseudomonas, Enterobacter, and Pantoea, while Alternaria and Fusarium were the dominant fungal groups. Although the microbiome structure changed substantially over time, there were no community-scale rearrangements due to the T6085 treatment. The work suggests several other taxa that could be explored as potential biocontrol agents to integrate with T6085 treatment. However, the timing and the type of T6085 application need to be improved to give more advantages for T6085 to colonize and reduce the F. graminearum inoculum in the field.

Insights on KP4 Killer Toxin-like Proteins of Fusarium Species in Interspecific Interactions.

KP4 killer toxins are secreted proteins that inhibit cell growth and induce cell death in target organisms. In Fusarium graminearum, KP4-like (KP4L) proteins contribute to fungal virulence in wheat seedling rot and are expressed during Fusarium head blight development. However, fungal KP4L proteins are also hypothesized to support fungal antagonism by permeabilizing cell walls of competing fungi to enable penetration of toxic compounds. Here, we report the differential expression patterns of F. graminearum KP4L genes (Fgkp4l-1, -2, -3 and -4) in a competitive interaction, using Trichoderma gamsii as the antagonist. The results from dual cultures indicate that Fgkp4l-3 and Fgkp4l-4 could participate in the recognition at the distance of the antagonist, while all Fgkp4l genes were highly activated in the pathogen during the physical interaction of both fungi. Only Fgkp4l-4 was up-regulated during the interaction with T. gamsii in wheat spikes. This suggests the KP4L proteins could participate in supporting F. graminearum interspecific interactions, even in living plant tissues. The distribution of KP4L orthologous within the genus Fusarium revealed they are more represented in species with broad host-plant range than in host-specific species. Phylogeny inferred provides evidence that KP4L genes evolved through gene duplications, gene loss and sequence diversification in the genus Fusarium.

Trichoderma-Induced Resistance to Botrytis cinerea in Solanum Species: A Meta-Analysis.

With the idea of summarizing the outcomes of studies focusing on the resistance induced by Trichoderma spp. against Botrytis cinerea in tomato, the present paper shows, for the first time, results of a meta-analysis performed on studies published from 2010 to 2021 concerning the cross-talk occurring in the tomato-Trichoderma-B. cinerea system. Starting from an initial set of 40 papers, the analysis was performed on 15 works and included nine parameters, as a result of a stringent selection mainly based on the availability of more than one article including the same indicator. The resulting work not only emphasizes the beneficial effects of Trichoderma in the control of grey mold in tomato leaves (reduction in disease intensity, severity and incidence and modulation of resistance genes in the host), but carefully drives the readers to reply to two questions: (i) What are the overall effects of Trichoderma on B. cinerea infection in tomato? (ii) Do the main effects of Trichoderma differ based on the tomato species, Trichoderma species, amount, type and duration of treatment? At the same time, this meta-analysis highlights some weak points of the available literature and should be seen as an invitation to improve future works to better the conceptualization and measure.

Complete Genome Sequence of the Plant-Pathogenic Fungus Colletotrichum lupini.

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fungal cross-talk: an integrated approach to study distance communication.

Despite the interest on fungi as eukaryotic model systems, the molecular mechanisms regulating the fungal non-self-recognition at a distance have not been studied so far. This paper investigates the molecular mechanisms regulating the cross-talk at a distance between two filamentous fungi, Trichoderma gamsii and Fusarium graminearum which establish a mycoparasitic interaction where T. gamsii and F. graminearum play the roles of mycoparasite and prey, respectively. In the present work, we use an integrated approach involving dual culture tests, comparative genomics and transcriptomics to investigate the fungal interaction before contact ('sensing phase'). Dual culture tests demonstrate that growth rate of F. graminearum accelerates in presence of T. gamsii at the sensing phase. T. gamsii up-regulates the expression of a ferric reductase involved in iron acquisition, while F. graminearum up-regulates the expression of genes coding for transmembrane transporters and killer toxins. At the same time, T. gamsii decreases the level of extracellular interaction by down-regulating genes coding for hydrolytic enzymes acting on fungal cell wall (chitinases). Given the importance of fungi as eukaryotic model systems and the ever-increasing genomic resources available, the integrated approach hereby presented can be applied to other interactions to deepen the knowledge on fungal communication at a distance.

Straw Competition and Wheat Root Endophytism of Trichoderma gamsii T6085 as Useful Traits in the Biological Control of Fusarium Head Blight.

Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing.

Isolation of Four Microalgal Strains From the Lake Massaciuccoli: Screening of Common Pollutants Tolerance Pattern and Perspectives for Their Use in Biotechnological Applications.

Aquatic ecosystems represent one of the largest reservoirs of phytoplankton accounting for most of the primary production of the Earth. The Lake Massaciuccoli located in Tuscany (Italy) is one of the largest swamps that in ancient times entirely covered the Versilia coastal plain. Despite its peculiar features, especially the eutrophic characteristics, its native microalgal consortia have never been explored up to now. In this work, we isolated and described four autochthonous microalgal strains from different sites in the lake (FB, Idr, CL_Sc, and CL_Ch); the four microalgal strains were identified within the Chlorella sorokiniana clade. We exposed them to ten of the most common or emerging environmental contaminants in order to describe their preliminary response to the tested substances: five metals (As, Fe, Ni, Cu, and Zn), two herbicides (Metolachlor and Sethoxydim), two antibiotics (Ciprofloxacin and Benzylpenicillin) and a non-steroidal anti-inflammatory drug (Ibuprofen). Physiological response of the strains highlighted intraspecific differences; strain CL_Sc was the most tolerant in presence of metals while strain Idr was the most sensitive. All strains were sensitive to sethoxydim and tolerant to metolachlor at all the tested concentrations. Strains FB and Idr were the most sensitive in presence of Ibuprofen while strain CL_Ch was the most sensitive to the highest Benzylpenicillin concentration. Resistance pattern of strain Idr somehow reflects both the phylogenetic and the geographic "isolation" from all other three strains. Finally, optical microscope observation confirmed some differences also in the microalgae morphological aspect. Overall, all the strains showed interesting responses in presence of high concentrations of the tested substances, representing putative interesting candidates for water remediation in wastewater treatment plants.

Combined Comparative Genomics and Gene Expression Analyses Provide Insights into the Terpene Synthases Inventory in Trichoderma.

Trichoderma is a fungal genus comprising species used as biocontrol agents in crop plant protection and with high value for industry. The beneficial effects of these species are supported by the secondary metabolites they produce. Terpenoid compounds are key players in the interaction of Trichoderma spp. with the environment and with their fungal and plant hosts; however, most of the terpene synthase (TS) genes involved in their biosynthesis have yet not been characterized. Here, we combined comparative genomics of TSs of 21 strains belonging to 17 Trichoderma spp., and gene expression studies on TSs using T. gamsii T6085 as a model. An overview of the diversity within the TS-gene family and the regulation of TS genes is provided. We identified 15 groups of TSs, and the presence of clade-specific enzymes revealed a variety of terpenoid chemotypes evolved to cover different ecological demands. We propose that functional differentiation of gene family members is the driver for the high number of TS genes found in the genomes of Trichoderma. Expression studies provide a picture in which different TS genes are regulated in many ways, which is a strong indication of different biological functions.

Genome Resources for the Endophytic Fungus Paraphaeosphaeria sporulosa.

Paraphaeosphaeria genus includes plant pathogens or biocontrol agents as well as bioremediators and endophytic fungi. Paraphaeosphaeria sporulosa 10515 was isolated in 2013 as an endophyte of Festuca spp. collected on Mount Etna at 1,832 meters above sea level. Here, we present the first-draft whole-genome sequence of a P. sporulosa endophytic isolate. This data will be useful for future research on understanding the genetic bases of endophytism.

Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae).

Blowfly, Lucilia sericata (Diptera: Calliphoridae), is a problematic synanthropic insect pest, a vector of microbial pathogens, and the causal agent of secondary myiasis. Fungal biopesticides are considered eco-friendly tools, alternative to synthetic pesticides, for the control of arthropod pests; however, to date, little is known about their bioactivity against blowflies. In this study, we assessed the insecticidal activity of three well-known entomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. sericata. In addition, we tested powdered carnauba wax as an electrically charged dust carrier in an attempt to enhance the virulence of fungal spores. Pathogenicity tests on adult flies, by adult immersion in conidial suspension (108 conidia mL-1), showed that the median lethal time (LT50) was 5.3, 5.9, and 6.2 days for B. bassiana, A. muscarius and B. pseudobassiana, respectively. In topical tests, when 108 dry conidia were mixed with or without carnauba wax, the LT50 was 7.7, 10.2, and 14 days without this carrier and 6.9, 8.6, and 13.8 days with it for B. bassiana, B. pseudobassiana and A. muscarius, respectively. Overall, our findings showed that, among the tested fungi, B. bassiana was the most virulent when formulated as a dry powder with carnauba wax, which greatly improved fungal efficacy against the blowfly. We discuss the utility of carnauba wax for electrostatic formulation powder of fungal spores in the integrated management of blowflies as an environmentally sustainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance.

Secondary metabolites produced by Colletotrichum lupini, the causal agent of anthachnose of lupin (Lupinus spp.).

COLLETOTRICHUM LUPINI: is the causal agent of lupin (Lupinus albus L.) anthracnose, a destructive seed-borne disease affecting stems and pods. Despite that several biological studies have been carried out on this pathogen, the production of secondary metabolites has not yet been investigated. Thus, a strain of C. lupini, obtained from symptomatic stems of L. albus, has been grown in vitro to evaluate its ability to produce bioactive compounds. From its culture filtrates, a 3-substituted indolinone, named lupindolinone, and a 5,6-disubstituted tetrahydro-α-pyrone, named lupinlactone, were isolated together with the known (3R)-mevalonolactone and tyrosol. Lupindolinone and lupinlactone were characterized as 3-ethylindolin-2-one and 5-hydroxy-6-methyltetrahydropyran-2-one by spectroscopic methods (essentially nuclear magnetic resonance [NMR] and high-resolution electrospray ionization mass spectrometry [HR ESI-MS]). The R absolute configuration (AC) at C-5 of lupinlactone was determined by applying the modified Mosher's method. Thus, considering its relative stereochemistry assigned by NMR spectroscopy, the AC of lupinlactone could be formulated as 5R,6S. Lupindolinone was isolated as racemic mixture as shown by investigation using chiroptical methods. The metabolites were assayed in different biological tests and proved to have some activities at the used concentration.

Use of Competitive Filamentous Fungi as an Alternative Approach for Mycotoxin Risk Reduction in Staple Cereals: State of Art and Future Perspectives.

Among plant fungal diseases, those affecting cereals represent a huge problem in terms of food security and safety. Cereals, such as maize and wheat, are very often targets of mycotoxigenic fungi. The limited availability of chemical plant protection products and physical methods to control mycotoxigenic fungi and to reduce food and feed mycotoxin contamination fosters alternative approaches, such as the use of beneficial fungi as an active ingredient of biological control products. Competitive interactions, including both exploitation and interference competition, between pathogenic and beneficial fungi, are generally recognized as mechanisms to control plant pathogens populations and to manage plant diseases. In the present review, two examples concerning the use of competitive beneficial filamentous fungi for the management of cereal diseases are discussed. The authors retrace the history of the well-established use of non-aflatoxigenic isolates of Aspergillus flavus to prevent aflatoxin contamination in maize and give an overview of the potential use of competitive beneficial filamentous fungi to manage Fusarium Head Blight on wheat and mitigate fusaria toxin contamination. Although important steps have been made towards the development of microorganisms as active ingredients of plant protection products, a reasoned revision of the registration rules is needed to significantly reduce the chemical based plant protection products in agriculture.